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US 10,059,990 B2Granted
In situ nucleic acid sequencing of expanded biological samples
AssigneeMassachusetts Institute of Technology
InventorsEdward Stuart Boyden, Fei Chen, Shahar Alon, George Church, Paul Warren Tillberg, Adam Marblestone, Evan Daugharthy
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Abstract
The invention provides in situ nucleic acid sequencing to be conducted in biological specimens that have been physically expanded. The invention leverages the techniques for expansion microscopy (ExM) to provide new methods for in situ sequencing of nucleic acids as well as new methods for fluorescent in situ sequencing (FISSEQ) in a new process referred to herein as “expansion sequencing” (ExSEQ).
Claims11
A method for in-situ sequencing of target nucleic acids present in a biological sample comprising the steps of:
- a) contacting the sample with a small molecule linker or a nucleic acid adaptor comprising a binding moiety and an anchor, wherein the binding moiety binds to target nucleic acids in the sample:
- and wherein the anchor comprises a polymerizable moiety;
- b) permeating the sample with a composition comprising precursors of a swellable material;
- c) initiating polymerization to form a swellable material wherein the swellable material is bound to the small molecule linker or a nucleic acid adaptor to form a sample-swellable material complex:
- d) digesting proteins present in the biological sample;
- e) adding an aqueous solvent or liquid to cause the sample-swellable material complex to swell thereby physically expanding the complex to form a first enlarged sample;
- f) re-embedding the first enlarged sample in a non-swellable polymer gel;
- g) modifying the target nucleic acids or the nucleic acid adaptor to form a nucleic acid adaptor useful for sequencing;
- and
- h) sequencing the nucleic acids present in the first enlarged sample.
7 more elements
Dependencies are inferred from the claim text.